The invention pertains to the field of molecular biology, in particular to synthesis of proteins and polypeptides in cell-free systems prepared from prokaryotic and eukaryotic cells.
Synthesis of polypeptides and proteins in cell-free translation systems of the first generation (U.S. Pat. No. 4,668,624, Roberts, 1978) was performed in a static (batch) mode where the reaction mixture is in static conditions with constant Mg2+, K+ and NTP concentrations, constant pH and temperature. To this end, extracts and lysates of prokaryotic (Zubay, 1973) and eukaryotic cells (Roberts and Paterson, 1973; Pelham and Jackson, 1976) were prepared, and natural and synthesized mRNAs were used (U.S. Pat. No. 4,937,190, Palmberg, 1990).
Rapid development of biotechnology has called for alternative methods that would increase the yield of synthesized proteins. The design of more productive translation systems in which the concentration of basic components is maintained constant during the synthesis is one direction of efforts aimed at improvement of the existing methods. In the second generation systems (Spirin et al., 1988), a continuous flow of low weight substrates included in the feeding solution (CFCF mode) into the reactor volume and removal of target polypetides and low molecular weight products inhibiting the cell-free system increases the time of its operation and raises the yield of the desired protein as compared to the classic system of synthesis in static (batch) conditions. Numerous studies have been focused on optimization of the conditions for CFCF protein synthesis (Baranov, 1989; Ryabova et al., 1989; Takanori et al., 1991; Spirin, 1992, Baranov and Spirin, 1993; Volyanik et al., 1993; Erdman et al., 1994; Kim and Choi, 1996; Yamamoto, 1996; Ryabova et al., 1998, EP Patent 0312617; Alakhov et al., 1993, EP Patent 0401369, Baranov et al., 1995, U.S. Pat. No. 5,434,079; Mozayeni, 1995; JP Patent 7075592, Shimizu, 1995; JP Patent 7031494, Sakurai, 1995; JP Patent 5076381, Sato, 1995; EP Patent 0593757, Baranov et al., 1997; U.S. Pat. No. 5,593,856, Choi et al., 1997).
U.S. Pat. No. 5,478,730 (Alakhov et al., 1995) describes a method in which the synthesis in cell-free translation systems is based on continuous exchange (CECF mode) of the feeding solution components with the component of the reaction mixture via a semipermeable barrier by a diffusion process. The results obtained by many authors (Davis et al., 1996; Kim and Choi, 1996; U.S. Pat. No. 5,593,856, Choi, 1997; JP Patent 10080295, Yamane, 1998) demonstrate a significant increase in the yield of the target polypeptide upon continuous exchange, as compared to the static (batch) mode of operation.
In addition to improvement of the components of the translation system, efforts were made to improve methods for preparation of mRNA in transcription systems including RNA polymerase and DNA. In these systems, preparation of mRNA depends on the concentration of RNA polymerase and DNA, as well as on the concentration of Mg2+, K+ and NTP and other ionic conditions (Kern and Davis, 1997). The cost of components of the in vitro transcription including RNA polymerase, DNA and NTP is very high. Therefore it is necessary to analyze conditions of transcription and optimize the process of mRNA preparation (Gurevich et al., 1991).
There are methods for synthesis of polypeptides in a CFCF mode in prokaryotic cell-free systems in conditions of a coupled transcription-translation (Baranov et al., 1989; EP Patent 0401369, Baranov et al., 1995; Ryabova et al., 1998) and the process was patented where transcription and translation occur in eukaryotic cell-free systems in the same reaction volume (Spirin, 1992; Baranov and Spirin, 1993; EP Patent 0593757, Baranov et al., 1997).
It is known (Craig et al., 1993) that translation and transcription conditions in eukaryotic cell-free systems differ and are determined largely by the concentrations of Mg2+ and K+. Therefore, two-stage (U.S. Pat. No. 5,665,563, Beckler, 1997; Operating Guide, Single Tube Protein(trademark), Novagen Inc., 1998) or three-stage synthesis (Roberts and Paterson, 1973) is widely used in a static (batch) mode. At the first stage optimal conditions are achieved for mRNA transcription, then the mRNA is purified or immediately added to a new reaction mixture with conditions for translation. A one-stage synthesis of polypeptides in a transcription-translation eukaryotic cell-free systems is known (U.S. Pat. No. 5,34,637, Thompson et al., 1994; Operation Guide, Linked in vitro SP6/T7 Transcription/Translation Kit, Roche Diagnostics GmbH, 1998). The authors of the patent (U.S. Pat. No. 5,324,637, Thompson et al., 1994) used a known principle of optimization of Mg2+ concentration in the reaction mixture. By adding Mg2+ to the reaction mixture prior to the synthesis, they achieved such a concentration of Mg2+ in the reaction system which is intermediate between the transcription optimum and the translation optimum. Further studies showed that such optimization has no advantages over the two-stage or three-stage procedures. The study of Laios et al. (1998) demonstrates that optimization of separate stages of transcription and translation is from 2 to 6 times more efficient than that of a coupled process. On the other hand, optimization of the selection of Mg2+ concentrations is based on a preliminary measurement of the magnesium concentration in the lysate or in the reaction volume which devalues the principle of the one-stage procedure.
European Patent 0593 757 (Baranov et al., 1997) describes the possibility to perform continuous CFCF synthesis of polypeptides in eukaryotic cell-free transcription-translation systems for 20 hours. During the synthesis, the Mg2+ concentration in the reaction mixture is maintained at the required level due to the constant concentration of Mg2+ in the feeding solution. Since ribonuclease activity in the reaction volume is low and the mRNA templates retain their activity for a prolonged time, the reaction system works with both the earlier and newly synthesized mRNA templates and synthesizes a target product due to the constant Mg2+ concentration. For a more productive synthesis, the transcription system should synthesize an adequate amount of mRNA. Therefore a large quantity of expensive polymerase SP6 or T7 (30,000 units) is required. It is mentioned in the text of the patent that optimal conditions of synthesis should be chosen in each individual case. To make an appropriate choice, it is necessary to perform a series of syntheses in a batch volume at different Mg2+ concentrations and determine its optimal value for the given polypeptide. Optimization of the process is time consuming and rather expensive.
There are many devices in which the continuous exchange mode (CECF) is maintained due to a diffusion process. The device, in the form of a dialysis container for synthesis of polypeptides in a cell-free system, was first described in U.S. Pat. No. 5,478,730 (Alakhov et al., 1995). Promega Corp. (Davis et al., 1996) made a comparative analysis of syntheses (in a static (batch) mode and at a continuous exchange mode) during coupled transcription-translation in a E. coli prokaryotic cell-free system. To this end, the authors used xe2x80x9cDispoDialyserxe2x80x9d instruments manufactured by Spectrum Medical Industr. (U.S. Pat. No. 5,324,428, Flaherty, 1994) and xe2x80x9cSlidealyzerxe2x80x9d dialysers manufactured by Pierce Chemical Comp. (U.S. Pat. No. 5,503,741, Clark. 1996).
For the synthesis of polypeptides upon coupled transcription-translation in a preliminarily concentrated prokaryotic cell-free system of E. coli, Kim and Choi (1996) used a dialysis membrane fixed at the bottom of a cylinder.
Yamamoto (1996) constructed a dialyser in which the membrane is made from hollow fibers. The feeding solution passes through the hollow fibers. Due to diffusion, the components of the reaction mixture exchange with those of the feeding solution.
In the device designed by Yamane (JP Patent 100809295, Yamane, 1998), the membrane is used to maintain constant conditions of synthesis due to diffusion of low molecular substrates of the feeding solution circulating along the dialysis membrane.
U.S. Pat. No. 5,478,730 (Alakhov et al., 1995) is most close to the dialyser operating in a continuous exchange mode. The authors of the patent give a detailed description of requirements for the porous barrier made either of a dialysis membrane, flat membrane or hollow fibers, which can be composed in multi-layered structures.
Many devices whose operation is based on a continuous flow (CFCF) mode have been developed. They differ from each other by the formation of the feeding solution flows and the modes of removal of products of synthesis and metabolism inhibiting operation of the system.
The use of one ultrafiltration membrane in a flow-type reactor is described in many papers (Spirin et al., 1988, 1992; Takanori et al., 1991; Spirin, 1992; Volyanik et al., 1993; Kim and Choi, 1996; Ryabova et al., 1998). A disadvantage of this method is that the incoming flow of the feeding solution is equal to the volume of the outcoming flow of low molecular and high molecular components resulting in fast closing of the pores of the ultrafiltration membrane.
In 1990 Fischer et al. (DE Patent 3914956) proposed a method using a multifold pulse supply of the feeding solution to the reaction volume. To this end, N cycles are formed to provide positive and negative pressure in the reaction volume. Upon formation of positive pressure, the inhibiting products are removed from the reaction volume via the porous barrier and mixed with the feeding solution. At negative pressure, part of the inhibiting products are returned to the reaction volume via the porous barrier together with another portion of the feeding solution. Moreover, high molecular weight components of the cell-free system required for a prolonged synthesis are intensely washed out from the reaction mixture.
In 1995 Mozayeny (U.S. Pat. No. 5,434,079) proposed a device with improved removal of high molecular weight products due to an increased area of the ultrafiltration membrane. During synthesis the components of the cell-free system are removed together with the target product via the large area of two parallel membranes with pore sizes from 70 to 100 kD, which limits the time of the synthesis.
The devices proposed herein are most close to the device described in U.S. Pat. No. 5,478,730 (Alakhov et al., 1995) with one or two porous barriers. The barriers can be made of flat membranes or hollow fibers.
Generally, the prior art describes methods and devices developed for maintaining constant conditions during the synthesis. Constant conditions are provided both by removal from the reaction volume the low molecular weight products which inhibit operation of the cell-free system and by supplying into the reaction volume some components which maintain the synthesis. The synthesis is maintained by the same concentrations of Mg2+, K+ and NTP and other components both in the reaction mixture and in the feeding solution. The authors of the prior art patent use a well known principle of optimization. Optimization of the process is time consuming and rather expensive.
The object of the present invention is to provide a method that will allow the synthesis of a target polypeptide in prokaryotic and eukaryotic cell-free systems. The invention is based on modification of methods of synthesis in a continuous flow (CFCF) mode or a continuous exchange (CECF) mode. In these modes, during the synthesis parallel to input into the reaction mixture of components maintaining the synthesis and output from the reaction mixture of low molecular weight components inhibiting the synthesis, concentrations of at least one of the selected components determining the productivity of the synthesis (Mg2+, K+, NTP, polyamines or their combinations) are continuously changed from the upper to the lower limit of the determined range.